Method for identifying modulators gene expression

ABSTRACT

A method of cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the micro-organism. It further relates to mammalian genes cloned by the present method, as well as to products encoded by such genes and antibodies which can bind the encoded proteins. More specifically, the present invention relates to a method of cloning mammalian genes which encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein, to products (RNA, proteins) enocded by the mammalian genes cloned in this manner and to antibodies which can bind the encoded proteins.

FUNDING

Work described herein was supported by the National Cancer Institute ofthe National Institutes of Health.

BACKGROUND OF THE INVENTION

Presently, there are several methods available for cloning mammaliangenes. The standard approach to cloning mammalian genes requiresobtaining purified protein, determining a partial amino acid sequence ofthe purified protein, using the partial amino acid sequence to producedegenerate oligonucleotide probes, and screening cDNA libraries withthese probes in order to obtain cDNA encoding the protein. This methodis time consuming and, because of the degeneracy of the probes used, mayidentify sequences other than those encoding the protein(s) of interest.Many mammalian genes have been cloned this way, including the cGMPphosphodiesterase expressed in retina (Ovchinnikov, Y-A. et al., FEB223: 169 (1987)).

A second approach to cloning genes encoding a protein of interest is touse a known gene as a probe to find homologs. This approach isparticularly useful when members of a gene family or families aresufficiently homologous. It is reasonable to expect that members of agiven gene family can be so cloned once one member of the family hasbeen cloned. The D. melanogaster dunce phosphodiesterase gene was used,for example to clone rat homologs. (Davis, R. L. et al., Proc. Natl.Acad. Sci. USA 86: 3604 (1989); Swinnen, J. V. et al., Proc. Natl. Acad.Sci. USA 86: 5325 (1989)). Although members of one family ofphosphodiesterase genes might be cloned once a member of that family hasbeen cloned, it is unclear whether the nucleotide sequences of genesbelonging to different phosphodiesterase gene families exhibitsufficient homology to use probes derived from one family to identifymembers of another family.

It would be useful to have a method which could be used to clone geneswhich does not have the limitations of presently available techniques.

SUMMARY OF THE INVENTION

The present invention relates to a method of cloning mammalian genesencoding proteins which can function in microorganisms, particularlyyeast, and can modify, complement, or suppress a genetic defectassociated with an identifiable phenotypic alteration or characteristicin the microorganism. It further relates to mammalian genes cloned bythe present method, as well as to products encoded by such genes andantibodies which can bind the encoded proteins. More specifically, thepresent invention relates to a method of cloning mammalian genes whichencode products which modify, complement or suppress a genetic defect ina biochemical pathway in which cAMP participates or in a biochemicalpathway which is controlled, directly or indirectly, by a RAS protein,to products (RNA, proteins) encoded by the mammalian genes cloned inthis manner and to antibodies which can bind the encoded proteins. Asdescribed herein, the present method has been used to identify novelmammalian genes which encode cAMP phosphodiesterases and proteins whichinteract with RAS proteins. These genes, and others that can be derivedby the claimed method, are part of this invention, as are the proteinswhich they encode.

The present invention further relates to a method of identifying agentswhich alter (i.e., reduce or stimulate) the activity of the proteinproducts of such mammalian genes expressed in microorganisms, such asyeast. Identification of such agents can be carried out using two typesof screening procedures: one based on biochemical assays of mammalianproteins of known enzymatic function and one based on phenotypic assaysfor proteins of unknown function. In the former case, if the encodedproteins are cAMP phosphodiesterases, pharmacological screens includethe assay for agents which alter (i.e., reduce or stimulate)phosphodiesterase activity. In the latter case, if the encoded proteinsinteract with RAS proteins, pharmacological screens include the assayfor agents which reduce or stimulate interactions with RAS proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of the steps of the present method of cloning amammalian gene which encodes a product capable of correcting a geneticalteration in a microorganism which is associated with an identifiablephenotypic characteristic.

FIG. 2 is a schematic representation of the yeast expression vectorsused to clone mammalian cDNAs. FIG. 2A is a schematic representation ofyeast expression vector pADNS. FIG. 2B is a schematic representation ofyeast expression vector pADANs. The origins of replication (ori, 2μ) andselectable markers (AmpR, LEU2) are shown, as are the alcoholdehydrogenase (ADH) promoter and terminator sequences. The polylinkerrestriction endonuclease sites are as shown. In FIG. 2B, the stippledarea indicates a portion of the ADH coding sequences.

FIG. 3 is the nucleotide sequence of DPD cDNA (top line) and its deducedamino acid sequence (bottom line). Nucleotide and amino acid coordinatesare given in the left hand margin.

FIG. 4 is the nucleotide sequence and the deduced amino acid sequence ofcDNA clone #44. Nucleotide and amino acid coordinates are given in theleft hand margin.

FIG. 5 is the nucleotide sequence and the deduced amino acid sequence ofcDNA clone #99. Nucleotide and amino acid coordinates are given in theleft hand margin.

FIG. 6 is the nucleotide and the deduced amino acid sequence of cDNAclone #265. Nucleotide and amino acid coordinates are given in the lefthand margin.

FIG. 7 is the nucleotide sequence and the deduced amino acid sequence ofcDNA clone #310. Nucleotide and amino acid coordinates are given in theleft hand margin.

FIG. 8 shows suppression of heat shock resistance resulting fromtreatment of yeast cultures with a pharmacological agent. Cells on theright plate were pretreated with 100 μM rolipram prior to heat shocktreatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of cloning a mammalian genewhich, when expressed in a microorganism, can modify, complement orsuppress, in the microorganism, a genetic alteration or defect which isassociated with an identifiable phenotype. Cloning of a selectedmammalian gene is carried out according to the present method byintroducing the gene into a genetically altered microorganism which hasan identifiable phenotypic alteration or characteristic associated withthe genetic alteration; maintaining the genetically alteredmicroorganism in which the gene is present under conditions appropriatefor cell growth; and selecting cells in which the phenotypic alterationor characteristic is modified as a result of correction, complementationor suppression of the genetic alteration or defect by the introducedmammalian gene. The present invention further relates to mammalian genescloned by the present method, products (e.g., RNA, proteins) encoded bysuch genes and antibodies specific for encoded proteins. In particular,the present invention relates to a method of cloning and isolating aselected mammalian gene which, when expressed in yeast having a geneticalteration or defect associated with an identifiable phenotypiccharacteristic or alteration, corrects, complements or supplements thegenetic alteration and modifies or corrects the associated phenotypiccharacteristic.

Through use of the method described herein, mammalian genes capable ofmodifying phenotypic alterations in yeast associated with activation orattenuation of biochemical pathways in which cAMP participates or withbiochemical pathways which interact with or are controlled by RASproteins have been identified, cloned and characterized. As describedherein, the subject method has been used to clone mammalian genesencoding cAMP phosphodiesterases or proteins which interact with a RASprotein. This has been accomplished using yeast cells in which there isa genetic alteration in the RAS2 gene and which, as a result, are heatshock sensitive. Additional mammalian genes having these samecharacteristics can be identified, cloned and characterized using themethod described. In each case, an appropriately-selected geneticallyaltered host cell (e.g., yeast) is used for expression of the selectedmammalian gene, which generally is introduced as one component of a genelibrary. The genetic alteration in the host cell is selected in such amanner that it is associated with an identifiable phenotypiccharacteristic which is corrected upon expression of the mammalian gene.The genetic alteration can be a deletion of a gene or a portion of agene, a change in nucleotide sequence or any other nucleotidemanipulation which renders a gene unable to function normally. Thegenetic alteration is selected in such a manner that a gene of interestcan be identified when it is expressed in the altered host cell.

The mammalian genes, when expressed in yeast containing a genetic defectin a biochemical pathway can correct, complement or alter the geneticdefect and correct the phenotypic alteration (i.e., produce a phenotypemore like that of normal or unaltered yeast). Cells containing themammalian gene to be cloned are identified on the basis of correction orsuppression of the phenotypic characteristic. The correction orsuppression need not be complete.

As described herein, it is now possible to identify inhibitors andactivators of cAMP phosphodiesterases, which can be used therapeuticallyto control or regulate cAMP levels or activity. In addition, it is nowpossible to identify agents which inhibit or stimulate interaction ofgene products with RAS proteins.

It is one of the objects of this invention to discover, isolate andcharacterize new genes encoding cAMP phosphodiesterases. It is a furtherobject of this invention to describe methods for identifying chemicalagents which inhibit or stimulate cAMP phosphodiesterases and can beused for therapeutic purposes. Both of these objectives can be achievedthrough use of methods, described herein, for cloning genes encodingcAMP phosphodiesterases and expressing the proteins that they encode incells with little or no other cAMP phosphodiesterase activity.Typically, cells used for expressing the proteins to be analyzed forcAMP phosphodiesterase activity lack other cAMP phosphodiesteraseactivity. Extracts from such cells thus provide a means by which agentswhich alter cAMP phosphodiesterases can be identified and isolated.

It is one of the further objects of this invention to discover, isolateand characterize new genes encoding products that interact with RASproteins. It is still a further object of this invention to describemethods for identifying agents which inhibit or stimulate theinteraction of these new gene products with RAS. This is accomplishedthrough use of methods described herein for cloning genes encodingproteins that interact with RAS, and expressing the encoded proteins incells that have phenotypes which are sensitive to the activity of theseproteins.

The following is a description of cAMP phosphodiesterases; pathwayscontrolled by RAS proteins; use of the present method for identificationof mammalian genes, exemplified by identification of genes encoding cAMPphosphodiesterases and genes encoding products which interact with RASproteins; use of the method to screen for agents which inhibit orstimulate cAMP phosphodiesterases; screening for agents which inhibit orstimulate interaction of such proteins with RAS proteins; and uses ofthe invention.

cAMP Phosphodiesterases

Adenylyl cyclase is an ubiquitous enzyme which generates cyclicadenosine monophosphate (cAMP). cAMP is a universal “second messenger”in both eukaryotes and prokaryotes. In eukaryotes, cAMP exerts itsprofound effects on cellular physiology by stimulating a cAMP-dependentprotein kinase (Robinson, G. A., et al., In Cyclic AMP, Academic Press(1971)). This kinase is composed of regulatory and catalytic subunits.The regulatory subunits combine with and inhibit the catalytic subunits.When the regulatory subunits bind cAMP, they release the catalyticsubunits, which in turn phosphorylate proteins on serine and threonineresidues. The genes encoding the catalytic and regulatory subunits havebeen cloned from yeast and mammals (Toda, T., et al., Cell 50: 277(1987); Shoji, S., et el., Biochemistry 22: 3702 (1983); Showers, M. O.and Mauver, R. A., J. Biol. Chem. 261: 16288 (1986); Titani, K., et al.,Biochemistry 23: 4193 (1984); Takio, K., et al., Biochemistry 23: 4200(1984)).

In mammals, cAMP is generated by cells in response to hormones, growthfactors, and neurotransmitters (Robison, G. A. et al., Cyclic AMP,Academic Press, N.Y. and London (1971)). The concentration of cAMP inmammalian cells is determined not only by its rate of production byadenylyl cyclase, but also by its rate of degradation by enzymes calledphosphodiesterases, or more specifically, cAMP phosphodiesterases.

A number of important physiological responses in humans are controlledby cAMP levels, including mental function, smooth muscle relaxation,strength of cardiac contractility, release of histamine and otherimmuno-reactive molecules, lymphocyte proliferation and plateletaggregation (Robison, G. A. et al., Cyclic AMP, Academic Press, N.Y. andLondon (1971)). Thus, the range of diseases which can potentially beaffected by agents or pharmaceutical compounds which alter cAMP levelsinclude inflammatory processes (e.g., arthritis and asthma), heartfailure, smooth muscle cramps, high blood pressure, blood clotting,thrombosis, and mental disorders. One way to modulate cAMP levels incells is through the modulation of cAMP phosphodiesterase activity.

Many drugs which raise cAMP levels in various tissues are in common use.These drugs are useful in treating heart failure, asthma, depression,and thrombosis. Only a few of these drugs appear to work by inhibitingcAMP phosphodiesterases. The pharmaceutical industry has not beennotably successful at finding such drugs, since effective drug screenshave not been available. The reasons for this are set forth below.

Most tissues contain so many different isoforms of phosphodiesterasesthat drug screening based on inhibition of crude tissue extracts isunlikely to yield anything other than a broadly acting inhibitor ofphosphodiesterases. Broadly acting inhibitors of cAMPphosphodiesterases, such as theophylline, have many deleterious sideeffects. A few inhibitors are known which have narrow specificity. Suchinhibitors may have great potential utility because they can targetphosphodiesterases in one or a few tissue and cell types and thus have ahigher therapeutic index.

The yeast cAMP phosphodiesterase genes PDE1 and PDE2 were the firstphosphodiesterase genes cloned (Sass, P., et al., Proc. Natl. Acad. Sci.USA, 83:9303 (1986); Nikawa, J., et al., Mol. Cell. Biol., 7:3629(1987)). Comparison of the amino acid sequence of the yeastphosphodiesterases to the amino acid sequences of other eukaryoticphosphodiesterases reveals only limited sequence homology. PDE2 has veryslight sequence homology to the dunce phosphodiesterase of D.melanogaster and to two phosphodiesterases expressed in bovine heart andbrain (Charbonneau, H., et. al., Proc. Natl. Acad. Sci., 83: 9308(1986.)). PDE1 shares even less of this homology, but resembles to agreater extent a secreted form of phosphodiesterase found in D.discoidem (Nikawa, J., et al., Mol. Cell. Biol. 7: 3629 (1987)). Thus,there appear to be many diverse branches of cAMP phosphodiesterase genesin evolution.

Biochemical, serological and pharmacological studies strongly suggestthe existence of multiple families of cAMP phosphodiesterases in mammals(Beavo, J. A. Advances in Second Messenger and Phosphoprotein ResearchVol. 22 1 (1988)). Partial amino acid sequence data and recent nucleicacid sequence data confirm this (Charbonneau, H., et al., Proc. Natl.Acad. Sci., USA 83: 9308 (1986); Colicelli, J., et al., Proc. Natl.Acad. Sci. USA 86: 3566 (1989); Davis, R. L., et al., Proc. Natl. Acad.Sci. USA 86: 3604 (1989); Swinnen, J. V., et al., Proc. Natl. Acad. Sci.USA 86: 5325 (1989)).

The various known phosphodiesterases fall into several classes: (I)Ca⁺⁺/calmodulin dependent, (II) cGMP stimulated, (III) cGMP inhibited,(IV) high affinity cAMP, (V) cGMP and (VI) nonspecificphosphodiesterases. Each class may be made up of a family of relatedproteins. In some cases these related proteins may be encoded byseparate genes and in other cases they may arise from alternative genesplicing. Generally, a tissue expresses multiple classes ofphosphodiesterases, which, by their copurification and proteolyticdegradation, render biochemical analysis exceedingly difficult. Theanalysis of this complexity is aided somewhat by the availability of afew pharmacological agents which discriminate between different classesof phosphodiesterases, and, recently, by serological re-agents which candistinguish between families and sometimes between members of a family(Beavo, J. A. Advances in Second Messenger and Phosphoprotein ResearchVol. 22: 1-38 (1988)).

The classification of mammalian phosphodiesterases may not be complete,however. New families and types of activities may yet be discovered. Thegreat majority of the cAMP phosphodiesterase genes have not yet beencloned.

Pathway Controlled by RAS Proteins

The RAS genes were first discovered as the transforming principles ofthe Harvey and Kirsten murine sarcoma viruses (Ellis, R. W., et al.,Nature 292: 506 (1981)). The cellular homologs of the oncogenes ofHarvey and Kirsten murine sarcoma viruses (H-RAS and K-RAS) constitutetwo members of the RAS gene family (Shimizu, K et al., Proc. Natl. Acad.Sci. 80:2112 (1983)). A third member is N-RAS (Shimizu, K. et al., Proc.Natl. Acad. Sci. 80: 2112 (1983)). These genes are known as oncogenessince point mutations in RAS can result in genes capable of transformingnon-cancerous cells into cancerous cells (Tabin, C. J., et al., Nature300: 143 (1982); Reddy, E. P., et al., Nature 300: 149 (1982);Taparowsky, E., et al., Nature 300: 762 (1982);). Many tumor cellscontain RAS genes with such mutations (Capon, D. J., et al., Nature302:33 (1983); Capon, D. J., et al., Nature 304: 507 (1983); Shimizu, K.et al., Nature 304: 497 (1983); Taparowsky, E., et al., Cell 34: 581(1983); Taparowsky, E., et al. Nature 300: 762 (1982); Barbacid, M.,Ann. Rev. Biochem. 56: 779 (1987)).

Despite the importance of the RAS oncogenes to our understanding ofcancer, the function of RAS genes in mammals is not known. The RASproteins are small proteins (21,000 daltons in mammals) which bind GTPand GDP (Papageorge, A., et al., J. Virol. 44: 509 (1982)). The RASproteins hydrolyze GTP slowly; specific cellular proteins can acceleratethis process (McGrath, J. P., et al., Nature 310: 644 (1984); Trahey,M., et al., Science 238: 542 (1987)). RAS proteins bind to the innersurface of the plasma membrane (Willingham, M. C., et al., Cell 19: 1005(1980)) and undergo a complex covalent modification at their carboxytermini (Hancock, J. F., et al., Cell 57: 1167 (1989)). The crystalstructure of H-RAS is known (De Vos, A. M et al., Science 239: 888(1988)).

The yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2,that have structural and functional homology with mammalian RASoncogenes (Powers, S., et al., Cell 36: 607 (1984); Kataoka, T., et al.,Cell 40: 19 (1985) Defeo-Jones, D. et al., Sciene 228: 179 (1985); Dhar,R., et al., Nucl. Acids Res. 12: 3611 (1984)). Both RAS1 and RAS2 havebeen cloned from yeast plasmid libraries and the complete nucleotidesequence of their coding regions has been determined (Powers, S., etal., Cell 36: 607 (1984); DeFeo-Jones, D., et al., Nature 306: 707(1983)). The two genes encode proteins with nearly 90% identity to thefirst 80 amino acid positions of the mammalian RAS proteins, and nearly50% identity to the next 80 amino acid positions. Yeast RAS1 and RAS2proteins are more homologous to each other, with about 90% identity forthe first 180 positions. After this, at nearly the same position thatthe mammalian RAS proteins begin to diverge from each other, the twoyeast RAS proteins diverge radically. The yeast RAS proteins, likeproteins encoded by the mammalian genes, terminate with the sequencecysAAX, where A is an aliphatic amino acid, and X is the terminal aminoacid (Barbacid, M., Ann., Rev. Biochem. 56: 779 (1987)). Monoclonalantibody directed against mammalian RAS proteins immumoprecipitates RASprotein in yeast cells (Powers, S., et al., Cell 47: 413 (1986)). Thus,the yeast RAS proteins have the same overall structure andinterrelationship as is found in the family of mammalian RAS proteins.

RAS genes have been detected in a wide variety of eukaryotic species,including Schizosaccharomyces pombe, Dictyostelium discoidem andDrosophila melanogaster (Fukui, Y., and Kaziro, Y., EMBO 4: 687 (1985);Reymond, C. D. et al., Cell 39: 141 (1984); Shilo, B-Z., and Weinberg,R. A., Proc. Natl. Acad. Sci., USA 78: 6789 (1981); Neuman-Silberberg,F., Cell 37: 1027 (1984)). The widespread distribution of RAS genes inevolution indicates that studies of RAS in simple eukaryotic organismsmay elucidate the normal cellular functions of RAS in mammals.

Extensive genetic analyses of the RAS1 and RAS2 of S. cerevisiae havebeen performed. By constructing in vitro RAS genes disrupted byselectable biochemical markers and introducing these by gene replacementinto the RAS chromosomal loci, it has been determined that neither RAS1nor RAS2 is, by itself, an essential gene. However, doubly RAS deficient(ras1⁻ ras2⁻) spores of doubly heterozygous diploids are incapable ofresuming vegetative growth. At least some RAS function is thereforerequired for viability in S. cerevisiae (Kataoka, T., et al., Cell 37:437 (1984)). It has also been determined that RAS1 is located onchromosome XV, 7 cM from ADE2 and 63 cM from HIS3; and that RAS2 islocated on chromosome XIV, 2 cM from MET4 (Kataoka, T., et al., Cell 37:437 (1984)).

Mammalian RAS expressed in yeast can function to correct the phenotypicdefects that otherwise would result from the loss of both RAS1 and RAS2(Kataoka, T., et al., Cell 40: 19 (1985)). Conversely, yeast RAS arecapable of functioning in vertebrate cells (De Feo-Jones, D., et al.,Science 228: 179 (1985)). Thus, there has been sufficient conservationof structure between yeast and human RAS proteins to allow each tofunction in heterologous host cells.

The missense mutant, RAS2^(va119), which encodes valine in place ofglycine at the nineteenth amino acid position, has the same sort ofmutation that is found in some oncogenic mutants of mammalian RAS genes(Tabin, C. J., et. al., Nature 300: 143 (1982); Reddy, E. P., et al.,Nature 300: 149 (1982); Taparowsky, E., et al., Nature 300: 762 (1982)).Diploid yeast cells that contain this mutation are incapable ofsporulating efficiently, even when they contain wild-type RAS alleles(Kataoka, T., et al., Cell 37: 437 (1984)). When an activated form ofthe RAS2 gene (e.g., RAS2^(va119)) is present in haploid cells, yeastcells fail to synthesize glycogen, are unable to arrest in G1, dierapidly upon nutrient starvation, and are acutely sensitive to heatshock (Toda, T., et al., Cell 40: 27 (1985); Sass, P., et al., Proc.Natl. Acad. Sci. 83: 9303 (1986)).

S. cerevisiae strains containing RAS2^(va119) have growth andbiochemical properties strikingly similar to yeast carrying the IAC orbcy1⁻ mutations, which activate the cAMP pathway in yeast (Uno, I., etal., J. Biol. Chem. 257: 14110 (1981)). Yeast strains carrying the IACmutation have elevated levels of adenylate cyclase activity. bcy1− cellslack the regulatory component of the cAMP dependent protein kinase (Uno,I. et al., J. Biol. Chem. 257: 14110 (1982); Toda, T., et al., Mol.Cell. Biol 7: 1371 (1987)). Yeast strains deficient in RAS functionexhibit properties similar to adenylate cyclase-deficient yeast (Toda,T., et al., Cell 40: 27 (1985)). The bcy1⁻ mutation suppresses lethalityin ras1⁻ 0 ras2⁻ yeast. These results suggest that in the yeast S.cerevisiae, RAS proteins function in the cAMP signalling pathway.

Adenylyl cyclase has been shown to be controlled by RAS proteins (Toda,T., et al., Cell 40: 27 (1985)). RAS proteins, either from yeast orhumans, can stimulate adenylyl cyclase up to fifty fold in in vitrobiochemical assays. RAS proteins will stimulate adenylyl cyclase onlywhen bound with GTP (Field, J., et al., Mol. Cell. Biol. 8: 2159(1988)).

The phenotypes which are due to activation of RAS, including sensitivityto heat shock and starvation, are primarily the result of overexpressionor uncontrolled activation of the cAMP effector pathway via adenylylcyclase (Kataoka, T., et al., Cell 37: 437 (1984); Kataoka, T. et al.,Cell 43: 493 (1985); Toda, T. et al., Cell 40: 27 (1985); Field J., etal., Mol. Cell. Biol., 8: 2159 (1988)). Two S. cerevisiae yeast genes,PDE1 and PDE2, which encode the low and high affinity cAMPphosphodiesterases, respectively, have been isolated (Sass, P., et al.,Proc. Natl. Acad. Sci. 83: 9303 (1986); Nikawa, J., et al., Mol. Cell.Biol. 7: 3629 (1987)). These genes were cloned from yeast genomiclibraries by their ability to suppress the heat shock sensitivity inyeast cells harboring an activated RAS2^(va119) gene. Cells lacking thePDE genes (i.e., pde1⁻ pde2⁻ yeast) are heat shock sensitive, aredeficient in glycogen accumulation, fail to grow on an acetate carbonsource, and in general have defects due to activation of the cAMPsignaling pathway (Nikawa, J., et al., Mol. Cell. Biol. 7: 3629 (1987)).

Genetic analysis clearly indicates that RAS proteins have otherfunctions in S. cerevisiae besides stimulating adenylyl cyclase (Toda,T. et al., Japan Sci Soc. Press. Tokyo/VNU Sci. Press, pp. 253 (1987);Wigler, M. et al., Cold Spring Harbor Symposium, Vol. LIII 649 (1988);Michaeli, T., et al., EMBO 8: 3039 (1989)). The precise biochemicalnature of these functions is unknown. Experiments with other systems,such as S. pombe and Xenopus laevis oocytes, indicate that RASstimulation of adenylyl cyclase is not widespread in evolution(Birchmeier, C., et al., Cell 43: 615 (1985)). It is unlikely that RASstimulates adenylyl cyclase in mammals (Beckner, S. K. et al., Nature317: 71 (1985)).

Identification of Mammalian Genes, Exemplified by Genes Encoding cAMPPhosphodiesterases

The present method can be used to clone a mammalian gene of interestwhich functions in a microorganism which is genetically altered ordefective in a defined manner (an altered microorganism) to correct thegenetic alteration or defect and, as a result, modifies an identifiablephenotypic alteration or characteristic associated with the geneticalteration or defect (produces a phenotype more like that of normal orunaltered yeast). Although use of the present method to clone andidentify mammalian genes is described in detail in respect to cAMPphosphodiesterases and proteins which interact with RAS proteins, it canbe used to clone and identify other mammalian genes which function in anappropriately-selected altered microorganism to correct, complement orsupplement the genetic alteration and, as a result, correct theassociated phenotypic alteration.

In its most general form, the method of the present invention isrepresented in FIG. 1 and can be described as follows: A cDNA library ofmammalian mRNAs is produced, using known techniques. This library can bemade by cloning double stranded cDNA into an expression vector. The cDNAcan be prepared from a preexisting cDNA library, or it can be preparedby the reverse transcription of mRNA purified from a tissue or cell lineof choice, using standard procedures (Watson, C. J. and Jackson, J. F.In: DNA cloning, a practical approach, IRL Press Oxford (1984)). This isdescribed in greater detail in Example 1, in which cDNA was derived fromrat brain mRNA and in Example 2, in which the cDNA was derived from ahuman glioblastoma cell line, U1188MG.

The cDNA obtained is cloned into an expression vector capable ofexpressing mammalian cDNA inserts as mRNA which can be translated intoprotein in a host cell of choice. Any expression vector, such as pADNS,into which the cDNA can be inserted and subsequently expressed as mRNAwhich is translated in an appropriate altered host cell (e.g., alteredyeast) can be used. Vectors which have been used for this purpose aredescribed; see FIG. 2A, which is a schematic representation of theexpression vector TK161-R2V, whose use is described in Examples 1 and 2,and FIG. 2B, which is a schematic representation of the expressionvector pADNS, whose use is described in Example 2. In general, anexpression vector contains a transcriptional promoter specific for thehost cell into which the vector is introduced For example, the vectorused in Examples 1 and 2 contains promoters for expression in S.cerevisiae. The expressed mRNA may utilize the ATG of the cDNA insert asthe “start” codon (e.g., the vector of FIG. 2A) or may express the cDNAproduct as a fusion protein (e.g., the vector of FIG. 2B).

The cDNA library (present as cDNA inserts in a selected expressionvector) is introduced into a host cell of choice, which contains geneticalterations which cause the host cell to have an identifiable phenotypicalteration or abnormality associated with the genetic alteration. Thehost cell may be a eukaryotic microorganism, such as the yeast S.cerevisiae. Known methods, such as lithium acetate-inducedtransformation, are used to introduce the cDNA-containing expressionvector. The genetic alterations may lead to defects in the metabolicpathways controlled by the RAS proteins and the associated readilydiscernible phenotype may be sensitivity to heat shock or nitrogenstarvation, failure to synthesize normal amounts of glycogen, failure togrow on certain carbon sources, failure to sporulate, failure to mate,or other properties associated with defects in the pathways controlledby RAS proteins. For example, as described in Examples 1 and 2, thegenetic alteration can be the presence of the RAS2^(va119) gene. Yeastcontaining such an alteration exhibit heat shock sensitivity, which, asdescribed in Examples 1 and 2, can be overcome by expression ofmammalian genes. Other genetic alterations can be chosen, such asdisruptions of the PDE1 and PDE2 genes in S. cerevisiae or disruptionsof,.or the presence of an activated allele of, ras1 in S. pombe.Different genetic alterations in the host cell may be correctable bydifferent subsets of mammalian cDNA genes.

After introduction of the cDNA insert-containing expression vector, hostcells are maintained under conditions appropriate for host cell growth.Those host cells which have been corrected for their phenotypicalteration are selected and the mammalian gene which they express isrecovered (e.g., by transformation of E. coli with DNA isolated from thehost cell). The mammalian gene is isolated and can be sequenced and usedfor further analysis in a variety of ways. For example, the encodedprotein can be identified and expressed in cultured cells for use infurther processes.

The present method has been used, as described in Examples 1 and 2, toisolate new mammalian genes whose presence in yeast cells has resultedin correction of a phenotypic alteration associated with a geneticalteration (the presence of the RAS2^(va119) gene). The nucleotidesequences of these genes, as well as the amino acid sequence encoded byeach, are described in Examples 1 and 2, and are shown in FIGS. 3-7. Thegenes of FIGS. 3 and 4 are homologous to the D. melanogaster dunce gene.The gene of FIG. 3 has also been recently isolated by others using theD. melanogaster dunce gene as probe (Swinnen, J. V., et al., Proc. Natl.Acad. Sci. 86: 5325 (1989)).

Screening and Identification of Agents which Alter cAMP PhospodiesteraseActivity

In its most general form, the second part of the invention(pharmacological screening) is carried out as follows: It is possible toscreen for agents that reduce or stimulate the activity of any mammalianprotein whose presence or expression in an altered microbial host cellin which a genetic alteration is associated with an identifiablephenotypic alteration results in correction of the phenotypicalteration. Two types of screens are possible, and are illustrated inExamples 3 and 4.

The first type of pharmacological screen is applicable when themammalian gene encodes a protein of known and assayable biochemicalfunction. The mammalian gene is first expressed in a microbial host byutilizing an appropriate host expression vector of the type alreadydescribed. Extracts of host cells are prepared, using known techniques;the cells are disrupted and their cellular constituents released. Crudecellular extract or purified mammalian protein is assayed for the knownbiochemical function in the presence of agents, the effects of which onthe protein are to be assessed. In this manner, agents which inhibit orstimulate the activity of the mammalian protein can be identified.

This type of procedure can be carried out to analyze the effects ofselected agents on mammalian cAMP phosphodiesterases. For example ayeast strain lacking both endogenous PDE1 and PDE2 genes can be used asthe host cell, into which cDNA encoding mammalian cAMP phosphodiesteraseis introduced in an appropriate expression vector and expressed. Such ahost cell is particularly useful because there is no background cAMPphosphodiesterase activity (Colicelli, J., et al., Proc. Natl. Acad.Sci. USA 86:3599 (1989)) and hence activity of the mammalian enzyme canbe cleanly assayed even in crude cell extracts. This procedure isillustrated in Example 3, in which it is demonstrated that the enzymaticactivity of the rat DPD gene product is inhibited by the pharmacologicalagents Rolipram and R020 1724, but not by the pharmacological agenttheophylline.

The second type of pharmacological screen is applicable even when themammalian gene encodes a protein of unknown function, and, thus, cannotbe assayed by a biochemical activity. In this method, agents to betested are applied or introduced directly to the genetically alteredmicrobial host expressing the mammalian protein. Agents capable ofinhibiting the mammalian gene or gene product are identified by theirability to reverse the phenotype originally corrected by expression ofthe mammalian protein in the altered host.

This procedure has been used for mammalian cDNAs encoding cAMPphosphodiesterases and a yeast containing RAS2^(va119) as the hoststrain (see Example 4). This host is heat shock sensitive. When the ratDPD gene is introduced into the heat shock sensitive host and expressed,the host strain becomes resistant to heat shock. When the now-resistantcells are incubated in Rolipram, they become heat shock sensitive again,indicating that Rolipram inhibits the activity of the rat DPD geneproduct. This pharmacological screen does not require that the functionof the DPD gene product be known. This same approach can be applied tothe genes which are the products of Example 2.

Applications of the Present Method and Products

The present method is useful for cloning novel mammalian genes whichencode cAMP phosphodiesterases or proteins which interact with a RASprotein. As described, novel mammalian genes have been cloned, using thepresent method, and the amino acid sequence of the encoded protein hasbeen deduced. Other mammalian genes encoding additional cAMPphosphodiesterases or additional proteins which interact with a RASprotein can be cloned using the method described. All or a portion ofthe sequence of the mammlaian genes encoding cAMP phosphodiesterases canbe used as probes, in known techniques, to identify homologs and theproducts encoded by such assayed homologs as described herein for cAMPphosphodiesterase activity. Similarly, all or a portion of the mammaliangenes encoding products which interact with RAS proteins can be used toidentify homologs and the ability of the encoded proteins to interactwith RAS proteins assessed as described herein.

Alternatively, mammalian genes encoding other proteins which function inan altered microorganism to correct, complement or supplement thealtered or defective genetic activity can be cloned, using amicroorganism with an appropriately-selected alteration (e.g., a changein a different biochemical pathway) which is associated with anidentifiable phenotypic characteristic.

The present invention is also useful for identifying agents,particularly chemical compounds, which alter (reduce or stimulate) cAMPphosphodiesterase and, thus, affect cAMP activity (e.g., by causing morerapid cAMP breakdown or inhibiting cAMP breakdown and, thus, shorteningor prolonging the duration of cAMP activity, respectively). The presentmethod is also useful for identifying agents which alter (inhibit orenhance) the interaction of gene products with RAS proteins.

Antibodies specific for proteins encoded by the mammalian genes isolatedusing the present method can be produced, using known techniques. Suchantibodies may be polyclonal or monoclonal and can be used to identifycAMP phosphodiesterases or proteins which interact with RAS proteins(e.g., the same proteins as those encoded by the mammalian genes orproteins sufficiently similar to the encoded proteins that they arerecognized or bound by an antibody raised against the encoded proteins).

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE 1 Identification of a Mammalian Gene that Can Revert the HeatShock Sensitivity of RAS2^(va119) Yeast

Several yeast genes have been isolated which, when overexpressed onextrachromosomal yeast vectors, are capable of suppressing the heatshock sensitivity exhibited by the RAS2^(va119) expressing strainTK161-R2V (Sass, P., et al., Proc. Natl. Acad. Sci. USA 83 (1986);Nikawa, J., et al., Mol. Cell. Biol., 7:3629 (1987)). As described inthis example, mammalian genes that can function in yeast to renderRAS2^(va119) cells resistant to heat shock have now been isolated. A ratbrain cDNA library was produced and cloned into the yeast expressionvector, pADNS (FIG. 1A). Double stranded cDNAs were prepared and ligatedto NotI linkers, cleaved with NotI restriction enzyme, and cloned intopADNS at the NotI site situated between the alcohol dehydrogenasepromoter and termination sequences of the vector. The use of the rarecutting NotI obviated the need for restriction site methylases commonlyused in cDNA cloning.

Approximately 1.5×10⁵ independent cDNA inserts were contained in thelibrary, with an average insert size of 1.5 kbp. DNA prepared from thecDNA expression library was used to transform the RAS2^(va119) yeaststrain, TK161-R2V. 50,000 Leu⁺ transformants obtained were subsequentlytested for heat shock sensitivity. Only one transformant displayed heatshock resistance which was conditional upon retention of the expressionplasmid. A plasmid, pADPD, was isolated from this transformant and the2.17 kb NotI insert was analyzed by restriction site mapping andnucleotide sequencing (FIG. 2).

A large open reading frame of 562 codons was found. The first ATGappears at codon 46 and a protein which initiates at this codon wouldhave a predicted molecular weight of approximately 60 kDa. This gene isdesignated DPD. A search for similar sequences was performed by computeranalysis of sequence data banks, and the Drosophila melanogaster duncegene was found. The two genes would encode proteins with an 80% aminoacid identity, without the introduction of gaps, over a 252 amino acidregion located in the center of the rat DPD cDNA. The dunce gene hasbeen shown to encode a high affinity cAMP phosphodiesterase (Chen, C.,et al., Proc. Natl. Acad. Sci. USA 83:9313 (1986); Davis, R. L. andKiger, J. A. J. Cell Biol. 90:101 (1981); Walter, M. F. and Kiger, J. A.J. Neurosci. 4:494 (1984)).

In order to demonstrate that the sequences upstream and downstream ofthe large sequence identity region were in fact contiguous with thatregion in the mRNA, rather than artifacts of the method for cDNAcloning, the structure of the cloned cDNA was compared with thestructure of DPD cDNAs contained in an independently prepared, firststrand cDNA population obtained by reverse transcribing total rat brainpoly (A)⁺ RNA with an oligo dT primer. Oligonucleotide primerscomplementary to sequences located within the identity region, and tosequences near the 5′ or 3′ ends of the coding strand, were made. Usingeither the cloned DPD DNA or the total first strand cDNA material astemplate, polymerase chain reactions (PCR) were carried out using fourdifferent primer sets and the reaction products were analysed bypolyacrylamide gel electrophoresis. In each case, a fragment of thepredicted length was obtained using either of the template DNAs. Theband assignments were confirmed by cleavage with restrictionendonucleases having recognition sites within the amplified DNA product.Again, in each case, the primary PCR product obtained using eithersource of template yielded cleavage products of the predicted sizes. Theresults indicate that the sequence arrangement in the cloned cDNAfaithfully reflects the structure of the rat mRNA.

Expression and Characterization of the DPD Gene Product

S. cerevisiae encodes two cAMP phosphodiesterase genes, PDE1 and PDE2(Sass, P., et al., Proc. Natl. Acad. Sci. USA 83:9303 (1986); Nikawa,J., et al., Mol. Cell. Biol. 7:3629 (1987)). The S. cerevisiae strain10DAB carries disruptions of both of these genes. The resulting cAMPphosphodiesterase deficiency leads to elevated intracellular cAMP levelsand a heat shock sensitivity phenotype similar to that of strainsharboring the RAS2^(va119) allele (Nikawa, J., et al. Mol. Cell. Biol.7:3629 (1987). 10DAB cells were transformed with the DPD expressionplasmid, pADPD, and assayed for heat shock sensitivity. Expression ofthe rat DPD gene indeed rendered this host resistant to heat shock.

In order to analyse the biochemical properties of the DPD gene product,crude cell extracts were prepared from one liter cultures of 10DAB whichhad been transformed with either pADNS or pADPD. Phosphodiesteraseactivity assays were performed using cAMP as substrate. Control extracts(10DAB with pADNS) showed no cAMP phosphodiesterase activity. Resultswith the controls were unchanged when performed at 0° C. or in theabsence of Mg²⁺ and were comparable to results obtained when no extractwas added. These results indicate that there is no detectable backgroundphosphodiesterase activity in strain 10DAB.

In contrast, considerable cAMP phosphodiesterase activity was seen inthe 10DAB strain transformed with pADPD. The rate of cAMP hydrolysis incells containing DPD was measured as a function of cAMP concentration.The deduced Km for cAMP is 3.5 μM and the calculated Vmax is 1.1nmol/mg/min.

The assay conditions were varied in order to ascertain the cationpreferences of the enzyme and to determine the ability of calcium andcalmodulin to stimulate its activity. In these assays, Mn²⁺ can beutilized as well as Mg²⁺, and either cation in 1 mM final concentrationwas sufficient. Calcium/calmodulin was unable to stimulate the measuredphosphodiesterase activity in the extract. A parallel assay using beefheart phosphodiesterase (Boeringer Mannheim) yielded a 6.5 foldstimulation with the addition of calcium/calmodulin. Finally, no cGMPphosphodiesterase activity was detected in these assays. Beef heartphosphodiesterase was again used as a positive control. In addition,cGMP present in amounts 100 fold over substrate concentrations wasunable to inhibit cAMP phosphodiesterase activity.

Strains, Media, Transformations and Heat Shock

Escherichia coli strain HB101 was used for plasmid propagation andisolation, and strain SCS1 (Stratagene) was used for transformation andmaintenance of the cDNA library (Mandel, M., and Higa, A. J. Mol. Biol.53: 159 (1970); Hanahan, D. J. Mol. Biol. 166:557 (1983)). Saccharomycescerevisiae strain TK161-R2V (MAT a leu2 his3 ura3 trp1 ade8 can1RAS2^(va119)) (Toda, T. et al., Cell40:27 (1985) and strain 10DAB wereused. Strain 10DAB was created from a segregant of a diploid strainproduced by mating TS-1 (Kataoka, T., et al., Cell 40:19 (1985)) andDJ23-3C (Nikawa, J. I., et al., Genes and Development 1:931 (1987)). Thesegregant (MATα leu2 his3 ura3 ade8 pde1::LEU2 pde2::URA3 ras1::HIS3)was subsequently transformed with the 5.4 kbp XbaI pde1::ADE8 fragmentof pYT19DAB to yield strain 10DAB. Yeast cells were grown in either richmedium (YPD) or synthetic medium with appropriate auxotrophicsupplements (SC) (Mortimer, R. K. and Hawthorne, D. C. In: The Yeast,vol. 1 385 (1969)). Transformation of yeast cells was performed withlithium acetate (Ito, H., et al., J. Bacteriol., 153:163 (1983)). Heatshock experiments were performed by replica plating onto preheated SCplates which were maintained at 55° C. for 10 minutes, allowed to cool,and incubated at 30° C. for 24-48 hrs. Segregation analysis wasperformed by growing yeast transformants in YPD for 2-3 days, platingonto YPD plates, and replica plating onto YPD, SC-leucine (plasmidselection), and YPD heat shock plates.

Plasmids DNA Manipulations and Sequencing

Plasmid DNA from individual E. coli colonies was purified by standardprocedures (Holmes, D. S., and Quigley, M. Anal. Biochem 114 193 (1981);Katz, L., et al., J. Bacteriol. 114 477 (1973). Extrachromosomal DNA wasisolated from yeast as previously described (Nikawa, J. et al., Mol.Cell. Biol., 7:3629 (1987)). The plasmid pYT19DAB was constructed frompYT19 (Nikawa, J. et al., Mol. Cell. Biol., 7:3629 (1987)) by firstdeleting PDE1 sequences between the SmaI and BalI restriction sites toyield pYT19D. The 4 kbp BamHI fragment of the ADE8 gene was theninserted into the BamHI site of pYT19D to yield pTY19DAB. The cloningvector pADNS is based on the plasmid pAD1 previously described (Powers,S., et al., Cell 47:413 (1986)). pADNS consists of a 2.2 kbp BglII toHpal fragment containing the S. cerevisiae LEU2 gene from YEp213(Sherman, F., Fink, et al., Laboratory Course Manual for Methods inYeast Genetics, eds. Sherman, F., Fink, G. R. and Hicks, J. B., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1986)), a 1.6 kbpHpaI to HindIII fragment of the S. cerevisiae 2μ plasmid containing theorigin of replication, and a 2.1 kbp SspI to EcoRI fragment containingthe ampicillin resistance gene from the plasmid pUC18. It also containsa 1.5 kbp BamHI to HindIII fragment of the modified S. cerevisiaealcohol dehydrogenase (ADH1J) promoter (Bennetzen, J. L. and Hall. B. D.J. Biol. Chem. 257:3018 (1982); Ammerer, G. Meth. Enzymol. 101:192(1983)) and a 0.6 kbp HindIII to BamHI fragment containing the ADH1terminator sequences. The promoter and terminator sequences areseparated by a polylinker that contains the restriction endonucleasesites NotI, SacII, and SfiI between the existing HindIII and SacI sites.The oligonucleotides used to create these sites were5′-GGCCAAAAAGGCCGCGGCCGCA and 5′-TCGACCGGTTTTTCCGGCGCCGGCGTTCGA. Theplasmid pADPD is a pADNS-derived plasmid containing the 2.17 kbp DPDcDNA insert.

Sequencing was performed using the dideoxy chain termination method(Sanger, R., et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977); Biggin,M. D., et al., Proc. Natl. Acad. Sci. USA 80:3963 (1983)). Genalign wasused to align the DPD and dunce sequences (GENALIGN is a copyrightedsoftware product of IntelliGenetics, Inc.; developed by Dr. HugoMartinez). RNA was purified from Sprague-Dawley rat brains by publishedprocedures (Chirgwin, J. M. et al., Biochem. 18:5294 (1979); Lizardi, P.M. Methods Enzymol 96:24 (1983); Watson C. J. and Jackson J. F. In: DNAcloning, a practical aproach, IRL Press Oxford (1984)). cDNAs wereligated to the NotI linker oligonucleotides 5′-AAGCCGGCCGC and5′-GCGGCCGCTT. The cDNAs were cleaved with NotI and cloned into the NotIsite of pADNS using standard procedures.

Polymerase chain reactions (PCRs) were carried out in thermocycler(Perkin Elmer, Cetus) using a modification of published procedures(Saiki, R., et al., Science 239:487 (1988)). Reaction mixtures containedtemplate DNA (1 ng of cloned DNA, or 1 μg of total first strand cDNA),25 pmoles of oligonucleotide primers, 200 μM deoxyribonucleotidetriphosphates, 10 mM Tris HCl (Ph8.4), 50 mM KCl, 3 mM MgCl₂, and 0.01%(w/v) gelatin. The oligonucleotide primers used, as designated in FIG.4, were:

-   A, 5′-CACCCTGCTGACAAACCT⁴⁴;-   B, 5′-ATGGAGACGCTCGAGGAA¹⁵³;-   C, 5′-ATACGCCACATCAGAATG⁶⁷⁶;-   D, 5′-TACCAGAGTATGATTCCC¹⁴⁴⁹;-   E, 5′-GTGTCGATCAGAGACTTG⁶⁶⁸ and-   F, 5′-GCACACAGGTTGGCAGAC²⁰⁴⁸. The numbers indicate position    coordinates in FIG. 3. Primers C, E and F are non-coding strand    sequences. Thirty cycles (1.5 min at 94° C., 3 min at 55° C., and 7    min at 72° C.) were performed and the reaction products were    analysed by polyacrylamide gel electrophoresis.    Phosphodiesterase Assays

Yeast cells were grown at 30° C. for 36 hours in one liter cultures ofsynthetic media (SC-leucine). Cells were harvested and washed withbuffer C (20 mM MES, 0.1 mM MgCl₂, 0.1 mM EGTA, 1 mM β-mercaptoethanol),were resuspended in 30 ml buffer C with 50 μl 1M PMSF, and weredisrupted with a French press. The extracts were centrifuged at 1,600 gfor 10 min and the supernatants were spun at 18,000 g for 90 min (4°C.). The supernatant was assayed for phosphodiesterase activity (Sass,P., et al., Proc. Natl. Acad. Sci. USA 83:9303 (1986);. Nikawa, J., etal., Mol. Cell. Biol. 7:3629 (1987)). All the reactions containedTris-HCl (pH7.5) (100 mM), cell extract (50 μg protein/ml),5′-nucleotidase (Sigma, 20 ng/ml) and 10 MM Mg²⁺ (unless otherwisestated) and the indicated cyclic nucleotide concentrations. Assays forthe cGMP hydrolysis used 1.5 μM cGMP. Inhibition studies employed 5 μMcAMP in the presence of varying amounts of cGMP up to 500 μM. [³H]cAMPand [³H]cGMP were obtained from NEN (New England Nuclear). Reactionswere incubated for 10 min at 30° C. and stopped with 5× stop solution(250 mM EDTA, 25 mM AMP, 100 mMcAMP).

Discussion

Previous workers have cloned a mammalian gene in yeast using abiological screen (Lee, M. G. and Nurse, P. Nature 327:31 (1987)). Inthat case, a homolog to the cdc2 gene of S. pombe was cloned byscreening a cDNA library for complementation of cdc2 mutants. In thatlibrary, the cDNAs were inserted proximal to the SV40 early T antigenpromoter. In our work we have employed a library with mammalian cDNAsinserted into a yeast expression vector, proximal to a strong yeastpromoter. In addition, we have employed NotI linkers for cDNA cloning,which allows the convenient subcloning of an entire insert library fromone vector to another. We feel that this will be a generally usefulapproach for cloning genes from higher eukaryotes when functionalscreens are possible in yeast. This system is particularly useful forthe cloning of other cAMP phosphodiesterases from mammals. Theavailability of yeast strains totally lacking endogenous cAMPphosphodiesterase activity will also facilitate the biochemicalcharacterization of these new phosphodiesterases.

The mammalian DPD cDNA can encode a protein with a high degree of aminoacid sequence identity (80%) with the predicted D. melanogaster duncegene product over an extended region. The dunce gene has been shown toencode a high affinity cAMP phosphodiesterase required for normallearning and memory in flies (Chen, C., et al., Proc. Natl. Acad. Sci.USA 83:9313 (1986); Davis R. L. and Kiger, J. A. J. Cell Biol. 90:101(1981); Walter, M. F. and Kiger, J. A. J. Neurosci. 4:495 (1984)).Compared to the striking level of sequence identity between DPD anddunce, the sequence conservation among other known cAMPphosphodiesterases is scant (Charbonneau, H., et al., Proc. Natl. Acad.Sci. USA 83:9308 (1986)). Therefore the DPD-dunce homology in theconserved region represents more than a constraint on sequences requiredfor cAMP binding and hydrolysis, and suggests a conservation ofinteractions with other components.

Biochemical characterization of the DPD cDNA product expressed in yeastindicates that it is a high affinity cAMP specific phosphodiesterase, asis dunce (Davis, R. L. and Kiger, J. A. J. Cell. Biol. 90:101 (1981);Walter M. F. and Kiger, J. A. J. Neurosci. 4 (1984)). In addition, DPDactivity, as measured in our assays, is not stimulated by the presenceof calcium/calmodulin. This property is shared with dunce and isdistinct from some other phosphodiesterases (Beavo, J. A. In Advances insecond messenger and phosphorprotein research, eds. Greengard, P. andRobinson, G. A., Raven Press, N.Y. vol. 22 (1988)). The two proteins,DPD and dunce, thus appear to have similar biochemical characteristics.However, it should also be noted that DPD encodes a protein productwhich shows much less significant homology (35%) to dunce beyond thepreviously described highly conserved core region. These non-conservedsequences could result in an altered or refined function for thismammalian dunce homolog.

The DPD sequence encodes a methionine codon at position 46 and theestablished reading frame remains open through to position 563,resulting in a protein with a predicted molecular weight of 60 kDa. Thesame reading frame, however, is open beyond the 5′ end of the codingstrand (FIG. 2). At present, it is not known if the methionine codon atposition 46 is the initiating condon for the DPD protein. The codingsequence is interrupted by three closely spaced terminator codons.However, the established reading frame then remains open for anadditional 116 codons, followed by more terminator codons, apolyadenylation consensus signal and a polyadenine stretch. This 3′ openreading frame could be incorporated into another dunce-likephosphodiesterase through alternate splicing.

Davis et al., (Davis, R. L. et al., Proc. Natl. Acad. Sci. USA 86:3604(1989)) have also isolated a mammalian dunce homolog from a rat braincDNA library using standard nucleic acid hybridization techniques. Thegene which they describe is indeed similar to, through distinct from,the DPD cDNA described here. Within the highly conserved region, asdefined in this work, the predicted amino acid sequences of the two ratgenes are 93% identical. This homology falls off dramatically, however,in the flanking regions which show amino acid identities of 60%(upstream) and 30% (downstream) and require the use of sequence gaps foroptimum alignment.

EXAMPLE 2 Identification of a Human Gene that Can Revert the Heat ShockSensitivity of RAS2^(va119) Yeast

A cDNA library was constructed in λZAP using NotI linkers. In thisexample, the cDNA derived from mRNA purified from the human glioblastomacell line U118MG. Inserts from the λ vector were transferred into twoyeast expression vectors. One, pADNS, is as described before. The other,pADANS (see FIG. 2B), differs in that the mRNA expressed will direct thesynthesis of a fusion protein: an N terminal portion derived from thealcohol dehydrogenase protein and the remainder from the mammalian cDNAinsert. Thus, two mammalian cDNA expression libraries were constructed.

These libraries were screened, as in the previous example, for cDNAscapable of correcting the heat shock sensitivity of the S. cerevisiaehost TK161-R2V. Several cDNAs were isolated and analysed by sequencing.Four different cDNA genes were thereby discovered, and their sequencesare shown in FIGS. 4-7.

The gene of FIG. 4 (JC44) was shown by computer analysis to behomologous to the rat DPD gene. Biochemical analysis has proven thatJC44 encodes a cAMP phosphodiesterase. The other genes, called JC99,JC265, and JC310, show no significant homology to previously isolatedgenes.

The genes of FIGS. 3 and 4 were shown to be able to correct thephenotypic defects of pde1⁻ pde2⁻ yeast strains. The genes of FIGS. 5-7were unable to do so. Thus, it appears that the latter genes do notencode cAMP phosphodesterases. Rather, these genes encode proteins ofunknown function which appear to be able to correct phenotypic defectsin yeast with activated RAS proteins.

Materials and Methods

Procedures of Example 1 were followed throughout. Described here is theconstruction of the plasmid pADANS, shown in FIG. 2B. A PCR reaction wascarried out on the yeast ADH1 gene in pJD14 (Bennetzen, J. L. and Hall,B. D. J. Biol. Chem. 257:2018 (1982)). One oligonucleotide primer (5′TCTAAACCGTGGAATATT) was placed within the promoter region of the gene.The second primer (5′ GTCAAAGCTTCGTAGAAGATAACACC) was designed tohybridize within the coding region of the gene. This primer included 5′non-hybridizing sequence encoding a HindIII endonuclease recognitionsite. The PCR product was then purified digested with HindIII and EcoRVand ligated into the 8.0 kb HindIII and EcoRV (partial) digestedfragment of pADNS. The resulting plasmid, pADANS, contains the entireADH1 promoter and the first 14 amino acid codons of the ADH1 genefollowed by the HindIII and NotI restriction endonuclease sites.

EXAMPLE 3 Identification of Agents which Inhibit PhosphodiesteraseActivity

This example illustrates the use of the genes and cells described inExample 1 to identify chemical compounds which inhibit the activity of aknown enzyme, the rat. DPD phosphodiesterase. To test the efficiency ofknown inhibitory compounds, cell free extracts were made as described inPhosphodiesterase Assays. Yeast cells deficient in endogenousphosphodiesterase (10DAB), and expressing the rat DPD or yeast PDE2genes from the described expression vector, were used. One litercultures were harvested, washed in buffer C (20 mM MES/0.1 mN MgCl₂/0.1mM EGTA/1 mM 2-mercaptoethanol), resuspended in buffer C containing 1.5mM phenylmethylsulfonyl fluoride, and disrupted in a French press at 4°C. Cell extracts were clarified at 100 g for 10 minutes and at 18000 gfor 90 minutes. PDE activities were assayed as published (Saiki et al.,Science 239:487-491 (1988); Charbonneau et al., Proc. Natl. Acad. Sci.USA 83:9308-9312 (1986); Tempel et al., Proc. Natl. Acad. Sci. USA80:1482-1486 (1983)) in a reaction mix containing 50 μg of cellprotein/ml, 100 mM Tris (pH 7.5), 10 mM Mg⁺⁺, 5 μM cAMP, 5′-nucleotidaseand [³H] cAMP. Hydrolysed AMP was separated from cAMP using AG1-X8 resinfrom Bio Rad. About 10⁴ cpm were obtained for 10 minutes reactions andbackgrounds (phosphodiesterase deficient-yeast or no extract) were about300 cpm. The cytosolic fraction was assayed in the presence or absenceof inhibitory compounds. These assays measure the amount of adenosine 5′monophosphate (AMP) produced by phosphodiesterase-catalysed hydrolysisof adenosine 3′, 5′-cyclic adenosine monophosphate (cAMP). For eachextract the percent inhibition for various concentrations of knowninhibitors is given in Table 1. The percent inhibition represents thedecrease in phosphodiesterase activity relative to measurements made inthe absence of inhibitors. Rolipram, and the related compound R020 1724,were the most effective inhibitors of DPD activity. TABLE 1 Inhibitionof Phosphodiesterases by Chemicals Phospho- Concentration Inhibitiondiesterase Agent (μM) (%) PDE2 Theophylline 250 0.0 IBMX 250 0.0 R0201724 100 3.0 Rolipram 100 0.0 DPD-1 Theophylline 250 42. IBMX 250 87.R020 1724 0.1 35. 1.0 52. 10.0 79. 100.0 92. Rolipram 0.1 50. 1.0 72.10.0 92. 100.0 95.

This analysis can, of course, be extended to test new or relatedchemical compounds for their ability to inhibit DPD activity, or theactivity of another phosphodiesterase expressed in this system. Clearly,this form of analysis can also be extended to other genes cloned andexpressed in a similar manner, for which there is an assayable enzymaticactivity.

EXAMPLE 4 Identification of Agents which Inhibit Mammalian Proteins ofUnknown Function Expressed in Yeast

This example illustrates the use of the genes and methods described toidentify chemical compounds which inhibit the function of the encodedmammalian proteins expressed in yeast, even when the function of thatprotein is not known. 10DAB cells, which are phosphodiesterasedeficient, are sensitive to heat shock. As already discussed, when thesecells express DPD, they become resistant to heat shock. FIG. 8demonstrates the inhibition of DPD function in yeast cells assayed byheat shock survival. 10DAB cells expressing DPD were maintained in richmedium (YPD) for three days at stationary phase. These cultures werethen treated with rolipram, a known phosphodiesterase inhibitor, for 40minutes at a final concentration of 100 μM. Control cultures were nottreated with any inhibitor. These cultures were then heat shocked inglass tubes at 50° C. for 30 minutes. One microliter of each culture wasplated. As shown in FIG. 8, cultures treated with rolipram (right side)were much more sensitive to heat shock, reflecting an inhibition of DPDenzymatic function.

Similarly, the suppression of heat shock sensitivity in the RAS2^(va119)yeast strain (TK161-R2V) by DPD expression will also be inhibited bydrug treatment. In addition, any other phenotype which is dependent onDPD phosphodiesterase activity should be affected by the presence of theinhibitory drug. The effect of a drug or agent can be assessed asdescribed. Finally, in the most generalized case, inhibitory chemicalsfor proteins of unknown function, expressed from mammalian cDNAs inyeast, can be discovered in a similar way. This approach depends only onthe phenotype consequent to expression of the protein and not onknowledge of its function.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. A method of cloning, in a genetically altered microorganism, a mammalian gene which is capable of modifying a phenotypic alteration associated with a genetic alteration in the microorganism, comprising the steps of: a) providing mammalian cDNA in an expression vector capable of expressing the mammalian cDNA in the genetically altered microorganism; b) introducing the expression vector into the genetically altered microorganism, thereby producing genetically altered microorganisms containing the expression vector; c) maintaining genetically altered microorganisms containing the expression vector under conditions appropriate for growth of genetically altered microorganisms; and d) identifying genetically altered microorganisms in which the phenotypic alteration associated with the genetic alteration in the microorganism is modified.
 2. The method of claim 1 further comprising transforming E. coli with cDNA present in genetically altered microorganisms of step (d).
 3. A method of cloning a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: a) providing mammalian cDNA in an expression vector capable of expressing the mammalian gene in yeast; b) introducing the expression vector of step (a) into genetically altered yeast cells, thereby producing genetically altered yeast cells containing the expression vector; c) maintaining genetically altered yeast cells containing the expression vector under conditions appropriate for growth of genetically altered yeast cells; and d) identifying genetically altered yeast cells in which the phenotypic alteration associated with the genetic alteration in the yeast cell is modified.
 4. The method of claim 3 further comprising transforming E. coli with cDNA present in genetically altered yeast cells of step (d).
 5. The method of claim 3 wherein the genetic alteration is associated with activation or attenuation of a biochemical pathway in which cAMP participates.
 6. The method of claim 3 wherein the genetic alteration is a mutation in a gene encoding a RAS protein.
 7. The method-of claim 6 wherein the genetic alteration is an activated RAS2^(va119) gene of S. cerevisiae.
 8. The method of claim 7 wherein the phenotypic alteration associated with a genetic alteration is selected from the group consisting of: heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate and failure to sporulate.
 9. The method of claim 3 wherein the genetically altered yeast cells are genetically altered S. cerevisiae or genetically altered S. pombe.
 10. The method of claim 5 wherein the genetic alteration is a disruption of the PDE1 and of the PDE2 genes of S. cerevisiae.
 11. The method of claim 6 wherein the genetic alteration is a disruption of the ras1 gene of S. pombe or an activated allele of the ras1 gene of S. pombe.
 12. Isolated DNA encoding a cAMP phosphodiesterase obtained by the method of claim
 1. 13. Isolated DNA having the nucleotide sequence of FIG.
 4. 14. Isolated protein encoded by the DNA of claim
 13. 15. Isolated DNA having the nucleotide sequence of FIG.
 5. 16. Isolated protein encoded by the DNA of claim
 15. 17. Isolated DNA having the nucleotide sequence of FIG.
 6. 18. Isolated protein encoded by the DNA of claim
 17. 19. Isolated DNA having the nucleotide sequence of FIG.
 7. 20. Isolated protein encoded by the DNA of claim
 19. 21. Isolated protein encoded by a nucleotide sequence which hybridizes to DNA having a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence of FIG. 4; b) the nucleotide sequence of FIG. 5; c) the nucleotide sequence of FIG. 6; and d) the nucleotide sequence of FIG.
 7. 22. A method of identifying a chemical agent which inhibits a mammalian gene which, when expressed in a genetically altered microorganism, modifies a phenotypic alteration associated with a genetic alteration in the microorganism, comprising the steps of: a) expressing the mammalian gene in a genetically altered microorganism, thereby modifying the phenotypic alteration associated with the genetic alteration; b) contacting the genetically altered microorganism of step (a) with a chemical agent to be assayed, under conditions appropriate for phenotypic assay; and c) determining whether the phenotypic alteration associated with the genetic alteration modified in step (a) is reversed, wherein reversal of the phenotypic alteration is indicative of a chemical agent which inhibits the mammalian gene.
 23. A method of identifying a chemical agent which inhibits a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: a) expressing the mammalian gene in genetically altered yeast cells in which the product encoded by the mammalian gene is not expressed, thereby modifying the phenotypic alteration associated with the genetic alteration; b) contacting genetically altered yeast cells of step (a) with a chemical agent to be assayed, under conditions appropriate for phenotypic assay; and c) determining whether the phenotypic alteration associated with the genetic alteration modified in step (a) is reversed, wherein reversal of the phenotypic alteration is indicative of a chemical agent which inhibits the mammalian gene.
 24. The method of claim 23, wherein the genetic alteration is associated with activation or attenuation of a biochemical pathway in which cAMP participates.
 25. The method of claim 23, wherein the genetic alteration is a mutation in a gene encoding a RAS protein.
 26. The method of claim 25, wherein the genetic alteration is an activated RAS2^(va119) gene of S. cerevisiae.
 27. The method of claim 23 wherein the genetically altered yeast cells are genetically altered S. cerevisiae or genetically altered S. pombe.
 28. The method of claim 27 wherein the phenotypic alteration associated with a genetic alteration is selected from the group consisting of: heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate, failure to mate and failure to sporulate.
 29. The method of claim 24 wherein the genetic alteration is a disruption of the PDE1 and a disruption of the PDE2 genes of S. cerevisiae.
 30. The method of claim 25 wherein the genetic alteration is a disruption of the ras1 gene of S. pombe or an activated allele of the ras1 gene of S. pombe.
 31. A method of identifying a chemical agent which alters activity of a protein encoded by a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: a) expressing the protein encoded by the mammalian gene in genetically altered yeast cells in which the protein encoded by the mammalian gene is not expressed, thereby modifying the phenotypic alteration associated with the genetic alteration; b) obtaining from genetically altered yeast cells produced in step (a) protein encoded by the mammalian gene; c) combining protein encoded by the mammalian gene with a chemical agent to be assayed for its ability to alter activity of the protein encoded by the mammalian gene; and d) determining activity of the protein encoded by the mammalian gene in combination with the chemical agent. 